MicroRNAs (miRNAs) are an extensive family of~21 nt long non-coding RNAs that are expressed in a wide range of eukaryotes, including humans. Current evidence suggests that miRNAs play a key role in differentiation and development by inhibiting the translation of mRNAs bearing partially complementary target sites. This effect has been reproduced in human cells in this laboratory using model mRNA targets and authentic but over-expressed human miRNAs. MiRNAs are closely related to small interfering RNAs (siRNAs), non-coding ~21 nt RNAs that can be introduced into cells as synthetic RNAs or derived from short hairpin RNAs (shRNAs) transcribed from expression plasmids. Although siRNAs usually function by inducing the degradation of mRNAs bearing fully complementary target sites, recent evidence suggests that siRNAs and miRNAs may function via similar mechanisms. Because of the likely importance of miRNAs in regulating gene expression, and the potential of siRNAs as artificial regulators of gene expression, it is important to understand how human miRNAs are made and how they function, if for no other reason than to permit the optimization of gene inactivation strategies that depend on siRNAs. In specific aim 1, we will attempt to understand how the initial miRNA transcript is precisely processed to yield a single, ~21 nt mature mRNA. Specifically, we will define the RNA sequences or structures that mediate the specific nuclear excision of the ~70 nt pre-miRNA intermediate from this initial transcript, a processing event very recently proposed to be mediated by human RNAse III/drosha. Moreover, we will define the features of this pre-miRNA that mediate the subsequent, equally specific cytoplasmic excision of the mature miRNA by dicer. In specific aim 2, we will extend our initial data showing that Exportin 5 (Exp5) is required for nuclear export of both pre-miRNAs and shRNAs, and that Exp5 binds the former specifically, by defining the characteristics of pre-miRNAs that mediate Exp5 binding. Specific aim 3 focuses on understanding how miRNAs cooperate to inhibit mRNA translation while specific aim 4 seeks to understood how endogenous miRNA stability and function is impacted by introduction of exogenous siRNAs. Finally, in specific aim 5, we seek to extend our exciting initial data demonstrating inhibition of miRNA function by the adenovirus VA1 non-coding RNA by trying to define the underlying mechanism.